Clinical organ transplantation has become one of the major treatments for end-stage organ failure since the introduction of chronic immunosuppressive drugs in the mid 1980s. This success has brought about the secondary issue of limited human organ supply, which greatly limits the ability to provide organs to patients in need of transplants. One of the major approaches to solving this medical need is the utilization of alternative species as a source of organs (xenotransplantation). R. W Evans, in Xenotransplantation, J. L. Platt, Ed. (ASM Press, Washington, D.C., 2001), pp. 29-51, teaches that the pig is the primary alternative species due to ethical considerations, breeding characteristics, infectious disease concerns and its compatible size and physiology.
A major barrier to progress in pig-to-primate organ transplantation is the presence of terminal α(1,3) galactosyl (gal) epitopes on the surface of pig cells. Humans and Old World monkeys have lost the corresponding galactosyltransferase activity in the course of evolution and will produce preformed natural antibodies against the epitope that are responsible for hyperacute rejection of porcine organs. The temporary removal of recipient anti-gal antibodies through affinity adsorption and expression of complement regulators in transgenic pigs has allowed survival of transplanted pig organs beyond the hyperacute stage. However, D. Lambrigts, D. H. Sachs, D. K. S Cooper, Transplantation 66, 547 (1998), teaches that returning antibody and residual complement activity are likely to be responsible for the acute and delayed damage which severely limits organ survival, even in the presence of high levels of immunosuppressive drugs and other clinical intervention.
Attempts have also been made to prevent rejection by reducing expression of gal epitopes through genetic engineering of the donor animal. Unfortunately, C. Costa et al., FASEB J. 13, 1762 (1999), discloses that competitive inhibition of galtransferase in H-transferase transgenic pigs results in only partial reduction in epitope numbers. Similarly, S. Miyagawa et al., J. Biol. Chem. 276, 39310 (2001), teaches that attempts to block expression of gal epitopes in N-acetylglucosaminyltransferase III transgenic pigs also results in only partial reduction of gal epitopes numbers and fails to significantly extend graft survival in primate recipients. Given the large number of gal epitopes present on pig cells, it seems unlikely that any dominant transgenic approach of this nature can provide sufficient protection from anti-gal mediated damage.
A. D. Thall, P. Maly, J. B. Lowe, J. Biol. Chem. 270, 21,437 (1995), teaches that viable α(1,3) galactosyltransferase knockout mice can be produced using embryonic stem cell technology. K. L. McCreath et al., Nature 405, 1066 (2000), teaches that nuclear transfer (NT) technology can be used for locus specific modification of certain large animals, as demonstrated by the production of viable sheep using in vitro targeted somatic cells. K. W. Park et al., Anim. Biotech. 12:173-181 (2001), discloses successful cloning and production of transgenic pigs by nuclear transfer of genetically modified somatic cells. Gustafsson and Sachs, U.S. Pat. No. 6,153,428 (2000), discloses genetically modified porcine cells in vitro in which the α(1,3)-galactosyltransferase gene has been disrupted by homologous recombination. Unfortunately, Bondioli et al., Mol. Reproduc. Dev. 60: 189-195 (2001) reports that the attempt to use nuclear transfer technology to accomplish this in pigs in vivo has been unsuccessful.
Therefore, there is a need for a method to generate viable swine with targeted gene knockouts, for example, α(1,3)-galactosyltransferase knockout swine. Such transgenic swine would provide organs, tissues, and cells useful in xenotransplantation.